The cellular mechanisms involved in adrenocorticotropin (ACTH) release were studied in a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16). CRF, the natural stimulant of ACTH release, stimulates secretion through a cAMP-dependent mechanism. This was demonstrated by incorporating cAMP-dependent protein kinase inhibitor into AtT-20 cells using a liposome technique. This manipulation abolished CRF stimulated ACTH release. In contrast, K plus/minus and phorbol ester evoked hormone secretion was not affected by this treatment indicating that there are multiple intracellular pathways involved in regulating the release of ACTH. CRF and 8-bromo-cyclic AMP also increase the levels of proopiomelanocortin (POMC) m-RNA in AtT-20 cells. This effect is blocked by the protein kinase inhibitor indicating that CRF stimulates both ACTH release and synthesis through an activation of cAMP-dependent protein kinase. Somatostatin (SRIF) is a potent inhibitor of ACTH release from AtT-20 cells. This peptide can block adenylate cyclase activity so as to prevent CRF and forskolin from evoking hormone secretion. SRIF also lowers intracellular calcium levels. This effect is blocked by pertussis toxin suggesting that either it is mediated by a guanine nucleotide inhibitory protein (Ni) or pertussis toxin desensitizes the SRIF receptor. Receptor binding studies show, in fact, that pertussis toxin does desensitize the SRIF receptor. SRIF also inhibits 8-bromo-cAMP and K+ evoked ACTH release but does not affect the ability of these secretagogues to increase calcium mobilization. These data indicate that SRIF acts through multiple mechanisms to regulate ACTH secretion.